A period of very active mitochondrial replication and development occurs during the transition into lactation in the mammary gland which provides an excellent opportunity to study mitochondrial biogenesis in a mammalian system. The objective of this project is to study these development phases in the intact mouse mammary gland and ultimately extend the observations to the organ explant culture system. Quantitation of mito-DNA and studies of isotope incorporation into mito-DNA during various stages of the pregnancy-lactation cycle, although somewhat preliminary, support the hypothesis that the greatest period of mitochondrial replication occurs coincidentally with major epithelial cell proliferation. This appears to end a day or two after cessation of cell division. The DNA per organelle then remains the same as a further expansion of mitochondrial protein occurs giving a drop in the DNA/protein ratio. We have previously demonstrated that a significant increase in mammary mitochondrial protein and enzymatic activities occurs during the first few days of lactation subsequent to epithelial proliferation. We have now shown that this is the result of a major expansion of the mitochondrial inner membrane. Our evidence for this consists of behavior on sucrose and ficoll gradients, changes in relative activities of inner and outer membrane markers, and electron microscopy.